Inhibition of Four-and-a-Half LIM Domain Protein-2 Increases Survival, Migratory Capacity, and Paracrine Function of Human Early Outgrowth Cells Through Activation of the Sphingosine Kinase-1 Pathway

Abstract

Rationale: Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair. Objective: We investigated the role of FHL2 in the regulation of EOCs. Methods and Results: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell–derived factor-1–induced EOC migration through upregulation of αv/β3, αv/β5, and β2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2 −/− mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2 −/− versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2 −/− versus WT EOCs. Conclusions: Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.

Absence of Four-and-a-Half LIM Domain Protein 2 Decreases Atherosclerosis in ApoE −/− Mice

“…We have recently shown that FHL2 deficiency is protective, promoting early outgrowth cell function as well as endothelial cell survival and function. 18 In the present study, we demonstrate that the absence of FHL2 in myeloid and vascular cells reduces plaque development in the ApoE −/− atherosclerotic mouse model. Moreover, we provide evidence that this atheroprotective effect could be, at least in part, because of reduced adhesion molecule expression in endothelial cells and because of a change in the chemokine/chemokine receptor expression balance leading to decreased recruitment of 20 and macrophages.…”

“…This result is in line with our previous data showing an improved re-endothelialization capacity in FHL2 −/− mice related to enhanced viability and migration of EC. 18 In addition, we observed a lower adhesion capacity of monocytes incubated with ApoE/FHL2 −/− ECs in a flow chamber. Hence, lower adhesive properties of ECs may well mitigate monocyte recruitment in ApoE/ FHL2 −/− mice.…”

The genetics of congenital heart disease… understanding and improving long-term outcomes in congenital heart disease: a review for the general cardiologist and primary care physician

“…CMA is a more advanced technique used to analyze the entire coding and non-coding regions of a patient’s genome for CNV mutations. Currently, CMA analysis detects approximately 70% of CNV mutations (62). WES is the most comprehensive test currently available.…”

“…It evaluates a patient’s entire exome for point mutations and sometimes CNV mutations (not all testing protocols include CNV analysis). WES may miss up to 5% of specific gene mutations, but its sensitivity continues to improve with time (62). …”

FHL2 interacts with iASPP and impacts the biological functions of leukemia cells

“…FHL2 can function as adaptors to mediate protein-protein interactions because of the LIM domain. Besides, FHL2 shuttles between cytosol and nucleus, and plays important roles in the regulation of signal transduction [9–12], cell survival [13, 14], motility [15, 16] and adhesion [17, 18]. Interestingly, FHL2 is upregulated in many cancers compared with normal tissues, for example, human melanoma, breast cancer and pancreatic cancer [11, 19, 20].…”